Synchrotron Radiation micro-CT Research Articles

The dissemination of Chinese potash-rich faience: investigation of faience beads from the Wupu cemetery, Xinjiang

The Wupu cemetery, located in the Yizhou District, Hami City, Xinjiang Province, and in the eastern part of the southern foothills of the Tianshan Mountains, is remains from the late Bronze Age. In this study, four faience beads excavated from the Wupu cemetery were examined by stereomicroscopy, synchrotron radiation micro-CT, and electron microprobe analysis. The appearance and compositions of these faience beads are presented and discussed. The results show that these tubular faience beads, glazed by the application glazing method, are all potash-rich faience, consistent with the faience produced locally in China. The faience products from the Central Plains were disseminated to the Hami region of Xinjiang Province during the Western Zhou dynasty (1046 BC–771 BC), reflecting the spread of such faience to the west with the Zhou people mastering the technique of making potash-rich faience.

Synchrotron radiation micro-CT with phase contrast for high-temperature in-situ microstructural characterization of Al Sn composite phase change materials

Thanks to immiscibility of Al and Sn, AlSn binary alloys can act as composite Phase Change Materials. These materials can store and release latent heat reversibly exploiting the solid-liquid transition of Sn at about 230 °C, combining this function with the thermal stability and the high conductivity of Al. To understand phenomena occurring during thermal cycles at high temperature, micro-CT analysis with phase contrast imaging was applied in-situ to AlSn alloys produced with different powder metallurgy processes. Tests were carried out at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Qualitative and quantitative analysis on 3D reconstructions were conducted in Matlab environment. With respect to previous studies, in-situ analysis provided a deeper understanding of the microstructural development depending on powder metallurgy processes on the composite microstructure. The overall form stability of the systems during a thermal cycle was proved. Molten Sn leakage phenomena are limited and related to Sn-rich areas directly connected to surface.

Impact of Anti-Angiogenic Treatment on Bone Vascularization in a Murine Model of Breast Cancer Bone Metastasis Using Synchrotron Radiation Micro-CT.

Simple SummaryThis study shows for the first time that the use of synchrotron radiation microcomputed tomography (SR-µCT) with a contrast agent enables the simultaneous quantitative measurement of three-dimensional (3D) bone and blood vessel microstructures in a murine model of breast cancer bone metastasis. Our data suggests that the combined anti-angiogenic drug (AAD) treatment (Bevacizumab + Vatalanib) dampens tumor-induced trabecular bone resorption and affects the 3D structure of bone microvascularization. The combined AAD might offer superior treatment to inhibit breast cancer bone metastasis in a mouse model.Bone metastases are frequent complications of breast cancer, facilitating the development of anarchic vascularization and induce bone destruction. Therefore, anti-angiogenic drugs (AAD) have been tested as a therapeutic strategy for the treatment of breast cancer bone metastasis. However, the kinetics of skeletal vascularization in response to tumor invasion under AAD is still partially understood. Therefore, the aim of this study was to explore the effect of AAD on experimental bone metastasis by analyzing the three-dimensional (3D) bone vasculature during metastatic formation and progression. Seventy-three eight-week-old female mice were treated with AAD (bevacizumab, vatalanib, or a combination of both drugs) or the vehicle (placebo) one day after injection with breast cancer cells. Mice were sacrificed eight or 22 days after tumor cell inoculation (time points T1 and T2, respectively). Synchrotron radiation microcomputed tomography (SR-μCT) was used to image bone and blood vessels with a contrast agent. Hence, 3D-bone and vascular networks were simultaneously visualized and quantitatively analyzed. At T1, the trabecular bone volume fraction was significantly increased (p < 0.05) in the combined AAD-treatment group, compared to the placebo- and single AAD-treatment groups. At T2, only the bone vasculature was reduced in the combined AAD-treatment group (p < 0.05), as judged by measurement of the blood vessel thickness. Our data suggest that, at the early stage, combined AAD treatment dampens tumor-induced bone resorption with no detectable effects on bone vessel organization while, at a later stage, it affects the structure of bone microvascularization.

Correction: Model of excision of the lateral half of the spinal cord at the lower thoracic level for the needs of reconstructive neurosurgery and neurotransplantation

Corrections to the article: https://doi.org/10.25305/unj.234154 In the article by V.V. Medvedev et al., published in UNJ № 3 in 2021, the source number 92 from the reference list does not support the statement given in the appropriate place in the text. Instead, we offer the reader two other works that mention the presence of posterior median spinal artery in the adult rat - D. Mazensky et al. (2017) and O.U. Scremin (G. Paxinos, ed.; 2015, p. 1003, 1005). In most works on this topic (Z. Zhang et al., 2001; Y. Cao et al., 2015; P. Li et al., 2020) the dorsal median vein is considered as the median vessel of the posterior surface of the rat spinal cord, and as in humans, describe 2 parallel dorsal spinal arteries. At the same time, D. Mazensky et al. (2017), sharing the opinion of O.U. Scremin (2015), mention 3 dorsal spinal arteries of the rat, in particular the median one. Taking into account that, from our experience, damage to the median vessel of the posterior surface of the spinal cord is accompanied by its rapid edema and irrepversible deep deficit in the motor function of both hind limbs of the animal, we consider it necessary to draw the reader's attention to this feature of the anatomy of the spinal arteries of an adult rat. Medvediev VV, Abdallah IM, Draguntsova NG, Savosko SI, Vaslovych VV, Tsymbaliuk VI, Voitenko NV. [Model of spinal cord lateral hemi-excision at the lower thoracic level for the tasks of reconstructive and experimental neurosurgery]. Ukr Neurosurg J [Internet]. 2021 Sep 27 [cited 2021 Oct 11];27(3):33-5. Available from: http://theunj.org/article/view/234154 Cao Y, Wu T, Yuan Z, Li D, Ni S, Hu J, Lu H. Three-dimensional imaging of microvasculature in the rat spinal cord following injury. Sci Rep. 2015 Jul 29;5:12643. doi: 10.1038/srep12643. PMID: 26220842; PMCID: PMC4518284. Li P, Xu Y, Cao Y, Wu T. 3D Digital Anatomic Angioarchitecture of the Rat Spinal Cord: A Synchrotron Radiation Micro-CT Study. Front Neuroanat. 2020 Jul 22;14:41. doi: 10.3389/fnana.2020.00041. PMID: 32792915; PMCID: PMC7387706. Mazensky D, Flesarova S, Sulla I. Arterial Blood Supply to the Spinal Cord in Animal Models of Spinal Cord Injury. A Review. Anat Rec (Hoboken). 2017 Dec;300(12):2091-2106. doi: 10.1002/ar.23694. Epub 2017 Oct 13. PMID: 28972696. Paxinos G, editor. The rat nervous system. 4th ed., London: Elsevier; 2015. Scremin OU. Capter 31, Cerebral Vascular System; p. 985‒1011. Zhang Z, Nonaka H, Nagayama T, Hatori T, Ihara F, Zhang L, Akima M. Circulatory disturbance of rat spinal cord induced by occluding ligation of the dorsal spinal vein. Acta Neuropathol. 2001 Oct;102(4):335-8. doi: 10.1007/s004010100377. PMID: 11603808.

When the Utility of Micro-Computed Tomography Collides with Insect Sample Preparation: An Entomologist User Guide to Solve Post-Processing Issues and Achieve Optimal 3D Models

Many techniques are used today to study insect morphology, including light and electron microscopy. Most of them require to specifically prepare the sample, precluding its use for further investigation. In contrast, micro-CT allows a sample to be studied in a non-destructive and rapid process, even without specific treatments that might hinder the use of rare and hard-to-find species in nature. We used synchrotron radiation (SR) micro-CT and conventional micro-CT to prepare 3D reconstructions of Diptera, Coleoptera, and Hymenoptera species that had been processed with 4 common preparation procedures: critical-point drying, sputter-coating, resin embedding, and air-drying. Our results showed that it is possible to further utilize insect samples prepared with the aforementioned preparation techniques for the creation of 3D models. Specimens dried at the critical point showed the best results, allowing us to faithfully reconstruct both their external surface and their internal structures, while sputter-coated insects were the most troublesome for the 3D reconstruction procedure. Air-dried specimens were suitable for external morphological analyses, while anatomical investigation of soft internal organs was not possible due to their shrinking and collapsing. The sample included in resin allowed us to reconstruct and appreciate the external cuticle and the internal parts. In this work, we demonstrate that insect samples destined to different analyses can be used for new micro-CT studies, further deepening the possibility of state-of-the-art morphological analyses.

3D visualization and morphometric analysis of spinal motion segments and vascular networks: A synchrotron radiation-based micro-CT study in mice.

The structure of spinal motion segments and spinal vasculature is complicated. Visualizing the three-dimensional (3D) structure of the spine may provide guidance for spine surgery. However, conventional imaging techniques fail to simultaneously obtain 3D images of soft and hard tissues, and achieving such coimaging states of the spine and its vascular networks remains a challenge. Synchrotron radiation micro-CT (SRμCT) provides a relatively effective and novel method of acquiring detailed 3D information. In this study, specimens of the thoracic spine were obtained from six mice. SRμCT was employed to acquire 3D images of the structure, and histologic staining was performed for comparisons with the SRμCT images. The whole spinal motion segments and the spinal vascular network were simultaneously explored at high resolution. The mean thickness of the cartilaginous end plates (CEPs) and the volume of the intervertebral discs (IVDs) were calculated. The surface of the CEPs and the facet joint cartilage (FJC) were presented as heat maps, which allowed for direct visualization of the thickness distribution. Regional division revealed heterogeneity among the ventral, central, and dorsal parts of the CEPs and between the superior and inferior parts of the facet processes. Moreover, the connections and spatial morphology of the spinal vascular network were visualized. Our study indicates that SRμCT imaging is an ideal method for high-resolution visualization and 3D morphometric analysis of the whole spinal motion segments and spinal vascular network.

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach

Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.

Biphasic Calcium Phosphate Biomaterials: Stem Cell-Derived Osteoinduction or In Vivo Osteoconduction? Novel Insights in Maxillary Sinus Augmentation by Advanced Imaging.

Maxillary sinus augmentation is often necessary prior to implantology procedure, in particular in cases of atrophic posterior maxilla. In this context, bone substitute biomaterials made of biphasic calcium phosphates, produced by three-dimensional additive manufacturing were shown to be highly biocompatible with an efficient osteoconductivity, especially when combined with cell-based tissue engineering. Thus, in the present research, osteoinduction and osteoconduction properties of biphasic calcium-phosphate constructs made by direct rapid prototyping and engineered with ovine-derived amniotic epithelial cells or amniotic fluid cells were evaluated. More in details, this preclinical study was performed using adult sheep targeted to receive scaffold alone (CTR), oAFSMC, or oAEC engineered constructs. The grafted sinuses were explanted at 90 days and a cross-linked experimental approach based on Synchrotron Radiation microCT and histology analysis was performed on the complete set of samples. The study, performed taking into account the distance from native surrounding bone, demonstrated that no significant differences occurred in bone regeneration between oAEC-, oAFMSC-cultured, and Ctr samples and that there was a predominant action of the osteoconduction versus the stem cells osteo-induction. Indeed, it was proven that the newly formed bone amount and distribution decreased from the side of contact scaffold/native bone toward the bulk of the scaffold itself, with almost constant values of morphometric descriptors in volumes more than 1 mm from the border.

Globular structure of the hypermineralized tissue in human femoral neck

Bone becomes more fragile with ageing. Among many structural changes, a thin layer of highly mineralized and brittle tissue covers part of the external surface of the thin femoral neck cortex in older people and has been proposed to increase hip fragility. However, there have been very limited reports on this hypermineralized tissue in the femoral neck, especially on its ultrastructure. Such information is critical to understanding both the mineralization process and its contributions to hip fracture. Here, we use multiple advanced techniques to characterize the ultrastructure of the hypermineralized tissue in the neck across various length scales. Synchrotron radiation micro-CT found larger but less densely distributed cellular lacunae in hypermineralized tissue than in lamellar bone. When examined under FIB-SEM, the hypermineralized tissue was mainly composed of mineral globules with sizes varying from submicron to a few microns. Nano-sized channels were present within the mineral globules and oriented with the surrounding organic matrix. Transmission electron microscopy showed the apatite inside globules were poorly crystalline, while those at the boundaries between the globules had well-defined lattice structure with crystallinity similar to the apatite mineral in lamellar bone. No preferred mineral orientation was observed both inside each globule and at the boundaries. Collectively, we conclude based on these new observations that the hypermineralized tissue is non-lamellar and has less organized mineral, which may contribute to the high brittleness of the tissue.

3D Digital Anatomic Angioarchitecture of the Rat Spinal Cord: A Synchrotron Radiation Micro-CT Study.

Comprehensive analysis of 3D angioarchitecture within the intact rat spinal cord remains technically challenging due to its sophisticated anatomical properties. In this study, we aim to present a framework for ultrahigh-resolution digitalized mapping of the normal rat spinal cord angioarchitecture and to determine the physiological parameters using synchrotron radiation micro-CT (SRμCT). Male SD rats were used in this ex vivo study. After a proportional mixture of contrast agents perfusion, the intact spinal cord covered the cervical spinal from the upper of the 1st cervical vertebra to the 5th lumbar vertebra was harvested and cut into proper lengths within three distinct regions: Cervical 3–5 levels, Thoracic 10–12 levels, Lumbar 3–5 levels spinal cord and examined using SRμCT. This method enabled the replication of the complicated microvasculature network of the normal rat spinal cord at the ultrahigh-resolution level, allowing for the precise quantitative analysis of the vascular morphological difference among cervical, thoracic and lumbar spinal cord in a 3D manner. Apart from a series of delicate 3D digital anatomical maps of the rat spinal cord angioarchitecture ranging from the cervical and thoracic to the lumbar spinal cord were presented, the 3D reconstruction data of SRμCT made the 3D printing of the spinal cord targeted selected microvasculature reality, that possibly provided deep insight into the nature and role of spinal cord intricate angioarchitecture. Our data proposed a new approach to outline systematic visual and quantitative evaluations on the 3D arrangement of the entire hierarchical microvasculature of the normal rat spinal cord at ultrahigh resolution. The technique may have great potential and become useful for future research on the poorly understood nature and function of the neurovascular interaction, particularly to investigate their pathology changes in various models of neurovascular disease.

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia.

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (Pi ) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn-/- ) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn-/- mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn-/- mice were fed a high-Pi diet, serum Pi concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-Pi diet, potentially indicating a role for elevated inhibitory pyrophosphate (PPi ) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum Pi , immunohistochemistry for OPN on Hyp mice fed a high-Pi diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum Pi may be attributable to the well-known upregulation of mineralization-inhibiting OPN by Pi , and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic Pi -regulating factors. © 2020 American Society for Bone and Mineral Research.

36M-pixel synchrotron radiation micro-CT for whole secondary pulmonary lobule visualization from a large human lung specimen

A micro-CT system was developed using a 36M-pixel digital single-lens reflex camera as a cost-effective mode for large human lung specimen imaging. Scientific grade cameras used for biomedical x-ray imaging are much more expensive than consumer-grade cameras. During the past decade, advances in image sensor technology for consumer appliances have spurred the development of biomedical x-ray imaging systems using commercial digital single-lens reflex cameras fitted with high megapixel CMOS image sensors. This micro-CT system is highly specialized for visualizing whole secondary pulmonary lobules in a large human lung specimen. The secondary pulmonary lobule, a fundamental unit of the lung structure, reproduces the lung in miniature. The lung specimen is set in an acrylic cylindrical case of 36 mm diameter and 40 mm height. A field of view (FOV) of the micro-CT is 40.6 mm wide × 15.1 mm high with 3.07 μm pixel size using offset CT scanning for enlargement of the FOV. We constructed a 13,220 × 13,220 × 4912 voxel image with 3.07 μm isotropic voxel size for three-dimensional visualization of the whole secondary pulmonary lobule. Furthermore, synchrotron radiation has proved to be a powerful high-resolution imaging tool. This micro-CT system using a single-lens reflex camera and synchrotron radiation provides practical benefits of high-resolution and wide-field performance, but at low cost.

Evaluating the effect of Avastin on breast cancer angiogenesis using synchrotron radiation.

The visualization of microvasculature is an essential step in understanding the mechanisms underlying early vessel disorders involved in breast cancer and for developing effective therapeutic strategies. However, generating detailed and reproducible data using immunohistochemistry analysis of breast cancer angiogenesis has been difficult. To analyze the diversification of angiogenesis in the development of tumor growth and evaluate the anti-vascular effects of Avastin (bevacizumab), we used new X-ray microangiography and third-generation synchrotron radiation-based micro-computed tomography (SR micro-CT) technology. With these techniques, we were able to investigate the structures and density of microvessels in xenograft mouse models (n=24). Barium sulfate nanoparticles were injected into the left cardiac ventricle of the mice to allow the visualization of blood vessels. Three-dimensional structures of microvessels were displayed with a high spatial image resolution of 20-30 µm. The density of angiogenesis and the incidence of lung metastasis were significantly reduced in xenograft mouse models of breast cancer treated with Avastin compared with control groups. Also, the density of smaller vessels (diameter <50 µm) was significantly decreased in the Avastin-treated mice, while the density of larger vessels (diameter >100 µm) was not significantly changed. Avastin inhibited tumor growth and lung metastasis by reducing microvessels. Additionally, synchrotron radiation (SR) techniques are useful as an additional tool for more precise quantification of angiogenesis.

Influence of loading condition and anatomical location on human cortical bone linear micro-cracks

Human cortical bone fracture toughness depends on the anatomical locations under quasi-static loading. Recent results also showed that under fall-like loading, cortical bone fracture toughness is similar at different anatomical locations in the same donor. While cortical bone toughening mechanisms are known to be dependent on the tissue architecture under quasi-static loading, the fracture mechanisms during a fall are less studied. In the current study, the structural parameters of eight paired femoral diaphyses, femoral necks and radial diaphyses were mechanically tested under quasi-static and fall-like loading conditions (female donors, 70 ± 14 y.o., [50–91 y.o.]). Synchrotron radiation micro-CT imaging was used to quantify the amount of micro-cracks formed during loading. The volume fraction of these micro-cracks was significantly higher within the specimens loaded under a quasi-static condition than under a loading representative of a fall. Under fall-like loading, there was no difference in crack volume fraction between the different paired anatomical locations. This result shows that the micro-cracking toughening mechanism depends both on the anatomical location and on the loading condition.

Cortical porosity assessment in the distal radius: A comparison of HR-pQCT measures with Synchrotron-Radiation micro-CT-based measures.

To determine the agreement between cortical porosity derived from high resolution peripheral quantitative computed tomography (HR-pQCT) (via standard threshold, mean density and density inhomogeneity methods) and synchrotron radiation micro-CT (SR-μCT) derived porosity at the distal radius. We scanned 10 cadaveric radii (mean donor age: 79, SD 11 years) at the standard distal region using HR-pQCT and SR-μCT at voxel sizes of 82 μm and 17.7 μm, respectively. Common cortical regions were delineated for each specimen in both imaging modalities. HR-pQCT images were analyzed for cortical porosity using the following methods: Standard threshold, mean density, and density inhomogeneity (via recommended and optimized equations). We assessed agreement in porosity measures between HR-pQCT methods and SR-μCT by reporting predicted variance from linear regression and mean bias with limits of agreement (LOA). The standard threshold and mean density methods predicted 85% and 89% of variance and indicated underestimation (mean bias -9.1%, LOA -15.9% to -2.2%) and overestimation (10.4%, 4.6% to 16.2%) of porosity, respectively. The density inhomogeneity method with recommended equation predicted 89% of variance and mean bias of 14.9% (-4.3 to 34.2) with systematic over-estimation of porosity in more porous specimens. The density inhomogeneity method with optimized equation predicted 91% of variance without bias (0.0%, -5.3 to 5.2). HR-pQCT imaged porosity assessed with the density inhomogeneity method with optimized equation indicated the best agreement with SR-μCT derived porosity.

The disuse effect on canal network structure and oxygen supply in the cortical bones of rats.

In this study, based on the measurements of intracortical vascular canal structure, we investigated the disuse effect on local O2 supply in the cortical bones of growing rats. Hindlimb disuse was produced by unilateral sciatic neurectomy (SN) at 4weeks age. The canal network structures within tibial cortical bone were evaluated in 8- and 12-week-old rats undergoing SN or no treatment (control) by synchrotron radiation micro-CT. Additionally, we developed an intracortical network model by combining the imaged-based canal network with a bone matrix containing theoretical lacunar-canalicular network, and determined the distribution of O2 concentration in bone tissue numerically. In the control bone, canal network was reduced with growth, resulting in decreased blood flow and averaged O2 concentration and increased spatial heterogeneity in tissue O2 concentration. Disuse reduced the canal network, leading to a lower flow rate, lower average O2 concentration and higher heterogeneity of O2 concentrations. However, the rarefaction of the canal network with growth was smaller under the disuse condition, and accordingly, the flow rate, the average O2 concentration and the heterogeneity of O2 concentrations remained stable. In particular, although the fraction of the canal volume was smaller, the densities of canal segments and bifurcation points under disuse condition tended to be higher than those of the control bone. The heterogeneity of O2 concentration was lower. Our results indicated that the disuse may lead to more uniformity in the canal network structure and thereby uniform O2, possibly contributing to O2 supply efficiency.

Impacts of Diabetes and an SGLT2 Inhibitor on the Glomerular Number and Volume in db/db Mice, as Estimated by Synchrotron Radiation Micro-CT at SPring-8.

BackgroundRecent large-scale clinical studies demonstrate that sodium-glucose cotransporter 2 (SGLT2) inhibitors protect the diabetic kidney. However, clinical and animal studies have not shown the changes of the total glomeruli in the whole kidney treated with SGLT2 inhibitors.MethodsWe performed computed tomography (CT) imaging on mice using synchrotron radiation to investigate the impact of luseogliflozin, a SGLT2 inhibitor, on the number and volume of glomeruli in the whole kidney.FindingsWe did not observe a significant difference in the total glomerular number (Nglom) among mice. Luseogliflozin redistributed the number of glomeruli in different regions, accompanied by the normalization of diabetes-augmented renal volume (Vkidney). Diabetic db/db mice had a larger glomerular volume in the mid-cortex than did control db/m mice, and luseogliflozin increased the glomerular volume in all renal cortical zones of the whole kidney in db/db mice. According to the multivariate regression analysis, hemoglobin A1c level was the most relevant determinant of Vkidney, not Nglom or mean glomerular volume (Vglom), indicating that hyperglycemia induced renal (tubular) hypertrophy, but not glomerular enlargement. Luseogliflozin increased hypoxia in the juxtamedullary region, sustained upregulated renal renin expression and plasma renin activity, and failed to decrease albuminuria by downregulating megalin in db/db mice.InterpretationBased on our findings, SGLT2 inhibitors may alter glomerular distribution and size in addition to their glucose-lowering effects, presumably by affecting oxygen metabolism and humoral factors.FundFunding for this research was provided by The Japan Society for the Promotion of Science, the Japan Diabetes Foundation, and Asahikawa Medical University.

Impact of Diabetes and an SGLT2 Inhibitor on Glomerular Number and Volume in db/db Mice as Estimated by Synchrotron Radiation Micro-CT at SPring-8

To investigate the impact of diabetes and luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor on the numbers and volumes of glomeruli in the whole kidneys, we performed CT imaging of diabetic db/db mice using synchrotron radiation at SPring-8. We found that there was no significant difference in the total glomerular number among mice. Diabetic mice had significantly larger glomerular volume in the mid-cortex compared to lean control db/m mice. Luseogliflozin reduced whole kidney volume, but not the glomerular hypertrophy in db/db mice. Luseogliflozin also decreased mesangial expansion and tubular damage in db/db mice. Luseogliflozin augmented hypoxia of S3 segment of proximal tubules in the juxtamedullary region and macula densa, as well as the number of intracellular vesicles in juxtaglomerular cells, and sustained upregulated renal renin expression in db/db mice. Luseogliflozin ameliorated hyperglycemia, glomerular and tubular injury, but not albuminuria. Db/db mice decreased the expression of renal megalin protein compared with that in db/m mice. Luseogliflozin-treated db/db mice also reduced the expression of megalin, accompanied with decreased reabsorbing urinary albumin as endocytotic ligand of megalin. In conclusion, our results suggest that oxygen metabolism and/or humoral factors more than blood glucose levels might determine the glomerular number and volume in diabetic kidney. Funding: This work was partially supported by a grant from the Japan Society for the Promotion of Science (JSPS KAKENHI grant no. 15K09372 to Y.T.), and by a Research Grant from the Japan Diabetes Foundation and Asahikawa Medical University (Research grant for Innovative Research in Life Science). Conflicts of Interests: Y.T. was also supported by several foundational grants, including grants from MSD, K.K., Eli Lilly and Company, Sanofi K.K., Takeda Pharmaceutical Co. and Taisho Pharmaceutical Co. No other potential conflicts of interest relevant to this article were reported. Ethical Approval Statement: These experiments were performed with the approval of the SPring-8 Proposal Review Committee (2012B1772, 2013A1655, 2013B1739).

Tetramethylpyrazine Facilitates Functional Recovery after Spinal Cord Injury by Inhibiting MMP2, MMP9, and Vascular Endothelial Cell Apoptosis.

Spinal cord injury (SCI) is a major public health issue that leads to neurological dysfunctions and morbidities in patients. Tetramethylpyrazine (TMP) plays a neuroprotective role in SCI; however, the underlying mechanism has not been fully elucidated. In the present study, we aimed to investigate the mechanisms and therapeutic effects of TMP on SCI. A contusion SCI model was established that used a modified Allen's method. In the TMP group, TMP (200 mg/kg) was injected daily for 5 days post-injury, while in the Negative Control (NC) group, an equal volume of normal saline was injected. Hindlimb motor function was evaluated using the Basso, Beattie, Bresnahan (BBB) scale. The effects of TMP on protein levels of the matrix metalloproteinases 2 (MMP2) and 9 (MMP9), Bax and cleaved caspase-3 were determined by western blotting. Apoptotic changes in vascular endothelial cells were evaluated using immunofluorescence and TUNEL staining. Alterations in 3D vessel morphology after treatment with TMP were assessed by synchrotron radiation micro-CT (SRμCT). TMP treatment significantly improved recovery in hindlimb motor function and attenuated vascular endothelial cell apoptosis in rats with SCI. Additionally, TMP treatment markedly decreased the protein levels of MMP2 and MMP9, pro-apoptotic bax and cleaved caspase-3 while promoting angiogenesis, as evidenced by vessel visualization using SRμCT. These results indicate that TMP attenuated SCI-induced neurological impairments by the down-regulation of the expression of MMP2 and MMP9 proteins, the inhibition of vascular endothelial cell apoptosis, and the promotion of angiogenesis.

Nondestructive characterization of ancient faience beads unearthed from Ya’er cemetery in Xinjiang, Early Iron Age China

Abstract Faience is a kind of ancient ceramic product which was widely found in many areas. Chinese faience appeared in the Western Zhou Dynasty (1046-771BCE), about three thousand years later than western Asia. The sudden and late emergence of Chinese faience implies that its production technology may be influenced by the West. However, little information is known about how the faience productions spread from the West to central China. The Xinjiang Uygur Autonomous Region, located in the northwestern part of China, is one of the most important areas of cultural exchanges between the East and the West since ancient times. The study of faience unearthed in Xinjiang is of great significance to explore the early trade and cultural exchanges between central China and the West. In this paper, Synchrotron Radiation Micro-CT (SR-μCT) and Energy dispersive X-ray fluorescence spectroscopy (EDXRF) have been applied to obtain the information of microstructure and chemical composition of faience beads excavated from Ya’er cemetery (1050-300BCE) in Hami Basin, The Xinjiang Uygur Autonomous Region, China. These faience beads are the earliest faience artifacts in China up to our knowledge and this is the first time to analyze the excavated faience in Xinjiang scientifically. Based on the microstructure of faience beads in CT slices, it is found that two glazing methods, direct application and efflorescence, were used in faience production. Additionally, a cylindrical core made of organic material sustained the faience bead during shaping and firing. According to EDXRF analysis, various glaze recipes have been used to produce these faience beads, possibly indicating different provenance. The characteristics of higher Na2O/K2O ratio of faience glaze showed that these faience beads might come from the West. Thus, it is suggested that there was a faience road from Western Asia through Xinjiang to central China about three thousand years ago.